Mass spectrometry (MS) coupled to affinity purification is a powerful approach for identifying protein-protein interactions and for mapping post-translational modifications. Prior to MS analysis, affinity-purified proteins are typically separated by gel electrophoresis, visualized with a protein stain, excised, and subjected to in-gel digestion. An inherent limitation of this series of steps is the loss of protein sample that occurs during gel processing. Although methods employing in-solution digestion have been reported, they generally suffer from poor reaction kinetics. In the present study, we demonstrate an application of a microfluidic processing device, termed the Proteomic Reactor, for enzymatic digestion of affinity-purified proteins for liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. Use of the Proteomic Reactor enabled the identification of numerous ubiquitinated proteins in a human cell line expressing reduced amounts of the ubiquitin-dependent chaperone, valosin-containing protein (VCP). The Proteomic Reactor is a novel technology that facilitates the analysis of affinity-purified proteins and has the potential to aid future biological studies.

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Keywords Affinity purification, H1299 cells, Mass spectrometry, Proteomic reactor, Ubiquitination, VCP
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Journal Journal of Proteome Research
Vasilescu, J. (Julian), Zweitzig, D.R. (Daniel R.), Denis, N.J. (Nicholas J.), Smith, J. C, Ethier, M. (Martin), Haines, D.S. (Dale S.), & Figeys, D. (Daniel). (2007). The proteomic reactor facilitates the analysis of affinity-purified proteins by mass spectrometry: Application for identifying ubiquitinated proteins in human cells. Journal of Proteome Research, 6(1), 298–305. doi:10.1021/pr060438j